Cellular zinc deficiency inhibits the mineralized nodule formation and downregulates bone-specific gene expression in osteoblastic MC3T3-E1 cells / 한국영양학회지

PURPOSE: Zinc (Zn) is an essential trace element for bone mineralization and osteoblast function. We examined the effects of Zn deficiency on osteoblast differentiation and mineralization in MC3T3-E1 cells. METHODS: Osteoblastic MC3T3-E1 cells were cultured at concentration of 1 to 15 µM ZnCl2 (Zn− or Zn+) for 5, 15 and 25 days up to the calcification period. Extracellular matrix mineralization was detected by staining Ca and P deposits using Alizarin Red and von Kossa stain respectively, and alkaline phosphatase (ALP) activity was detected by ALP staining and colorimetric method. RESULTS: Extracellular matrix mineralization was decreased in Zn deficiency over 5, 15, and 25 days. Similarly, staining of ALP activity as the sign of an osteoblast differentiation, was also decreased by Zn deficiency over the same period. Interestingly, the gene expression of bone-related markers (ALP, PTHR; parathyroid hormone receptor, OPN; osteopontin, OC; osteocalcin and COLI; collagen type I), and bone-specific transcription factor Runx2 were downregulated by Zn deficiency for 5 or 15 days, however, this was restored at 25 days. CONCLUSION: Our data suggests that Zn deficiency inhibits osteoblast differentiation by retarding bone marker gene expression and also inhibits bone mineralization by decreasing Ca/P deposition as well as ALP activity.

Study on mechanism of Drynariae Rhizoma in treating osteoporosis with integrative pharmacology perspective / 中国中药杂志

Drynariae Rhizoma has great significance in the clinical practice of osteoporosis treatment. Based on the perspective of integrative pharmacology, the study explored the mechanism of action of Drynariae Rhizoma in the treatment of osteoporosis. Six active components in Drynariae Rhizoma were obtained, mainly including glycosides and sterols. Taking the median of 2 times of "node connectivity" as the card value, the core node of the Chinese medicine target disease gene interaction network was selected. Based on this, three topological structural eigenvalues, such as "node connectivity" "node tightness" and "node connectivity" were calculated, thereby screening out four core targets of Drynariae Rhizoma treatment for osteoporosis, including thyroid parathyroid hormone 1 receptor (PTH1R), parathyroid hormone 2 receptor (PTH2R), calcitonin receptor gene (CALCR), and SPTBN1 gene (SPTBN1). Based on the gene ontology database GO and KEGG pathway database, the molecular function, intracellular localization, and biological reactions and pathways of proteins encoded by drug target genes were determined. Combined with enrichment calculation, it is predicted that osteoporosis may play a role in biosynthetic processes, such as circulatory system, nervous system, energy metabolism, prolactin signal pathway, GnRH signaling pathway, neurotrophic factor signaling pathway and other pathway. The conclusion of this study is certain with the existing research results, and the new target and new pathway could also be used as a theoretical basis for the further verification of osteoporosis.

Effects of experimentally created unilateral anterior crossbite prosthesis on the expression of parathyroid hormone-related peptide and parathyroid hormone receptor-1 in mandibular condylar cartilage of rat / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the effects of experimentally created unilateral anterior crossbite prosthesis on the expression of parathyroid hormone-related peptide (PTHrP) and parathyroid hormone receptor-1 (PTH1R) in mandibular condylar cartilage of SD rat.</p><p><b>METHODS</b>In experimental groups, the unilateral anterior crossbite metal prosthesis was cemented to the left incisors of the maxilla and mandible of 6-week-old SD rats, respectively. Animals were sacrificed at 2, 4, 8 weeks. Hematoxylin-eosin (HE) staining were carried out for studying the morphological changes of the condylar cartilage. Immunohistochemical staining and real-time polymerase chain reaction (PCR) analysis were performed to detect the levels of expression of PTHrP and PTH1R in the condylar cartilages.</p><p><b>RESULTS</b>The obvious degenerative changes were found in the condylar cartilages in experimental group at 8 weeks. Comparing to the control group, the expression of PTHrP mRNA (P < 0.01) and protein(P < 0.01) in the experimental group were increased, whereas PTH1R mRNA (P < 0.01) and protein (P < 0.01) levels were decreased.</p><p><b>CONCLUSION</b>The expression of PTHrP was increased in the condylar cartilage of rat with unilateral anterior crossbite metal prosthesis but its effects might be limited because of decreased expression of PTH1R in the condylar cartilage. The low level expression of PTH1R should be a part of the constitution of the molecular pathomechanism of temporomandibular joint osteoarthritis (TMJOA)-like lesion.</p>

Design and activity verification of human parathyroid hormone (1-34) mutant protein / 药学学报

Through protein-protein BLAST of homologous sequences in different species in NCBI database and preliminary simulating molecular docking and molecular dynamics by computer software discovery studio 3.1, three amino acids R25K26K27 of natural human parathyroid hormone (1-34) with Q25E26L27 were mutated and the biological activity of the mutant peptide was evaluated. Result showed that: root mean superposition deviation RMSD value between PTH (1-34)-(RKK-QEL) and PTH (1-34) peptide main chain was 2.509 3, indicating that the differences between the two main chain structural conformation was relatively small; the interaction energy between PTH (1-34)-(RKK-QEL) and its receptor protein PTH1R had been enhanced by 7.5% compared to nature PTH (1-34), from -554.083 kcal x mol(-1) to -599.253 kcal x mol(-1); the number of hydrogen bonds was increased from 32 to 38; PTH (1-34)-(RKK-QEL) can significantly stimulate the RANKL gene expression (P < 0.01) while inhibiting the OPG gene expression (P < 0.01) in UAMS-32P cells; in the co-culture system of UAMS-32P cells and mouse primary femur bone marrow cells, PTH (1-34)-(RKK-QEL) stimulated the formation of osteoclasts (P < 0.01) and had a higher biological activity than PTH (1-34) standard reagents.

Construction and identification of siRNA recombinant expression vector targeting parathyroid hormone 1 receptor gene and its effect on the cell cycle of INS-1 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct the expression vector of siRNA targeting parathyroid hormone 1 receptor (PTH1R) gene and evaluate its effect on the cell cycle of INS-1 cells.</p><p><b>METHODS</b>The sequences of PTH1R gene was retrieved from Genbank, and 4 pairs of oligonucleotides were synthesized and inserted into pSUPERretro RNAi, which was identified by RT-PCR and sequence analysis. The vectors were then transfected into INS-1 cells, in which the expression of PTH1R was observed by Western blotting to evaluate the transfection efficiency. The cell cycle of INS-1 cells in high glucose medium was detected by flow cytometry.</p><p><b>RESULTS</b>RT-PCR and sequence analysis confirmed the correct construction of the siRNA recombinant expression vector targeting PTH1R gene. The vectors were successfully transfected into INS-1 cells, and the most effective vector was selected by Western blotting. Transfection with the siRNA for PTH1R gene silencing resulted in the inhibition of INS-1 form entering the S phase.</p><p><b>CONCLUSION</b>The successful construction of the recombinant PTH1R-siRNA vectors establishes a basis for further study of protective role of the PTH1R gene in INS-1 cells in high glucose medium.</p>

The N- and C-terminal domains of parathyroid hormone-related protein affect differently the osteogenic and adipogenic potential of human mesenchymal stem cells

Parathyroid hormone-related protein (PTHrP) is synthesized by diverse tissues, and its processing produces several fragments, each with apparently distinct autocrine and paracrine bioactivities. In bone, PTHrP appears to modulate bone formation in part through promoting osteoblast differentiation. The putative effect of PTH-like and PTH-unrelated fragments of PTHrP on human mesenchymal stem cell (MSCs) is not well known. Human MSCs were treated with PTHrP (1-36) or PTHrP (107-139) or both (each at 10 nM) in osteogenic or adipogenic medium, from the start or after 6 days of exposure to the corresponding medium, and the expression of several osteoblastogenic and adipogenic markers was analyzed. PTHrP (1-36) inhibited adipogenesis in MSCs and favoured the expression of osteogenic early markers. The opposite was observed with treatment of MSCs with PTHrP (107-139). Moreover, inhibition of the adipogenic differentiation by PTHrP (1-36) prevailed in the presence of PTHrP (107-139). The PTH/PTHrP type 1 receptor (PTH1R) gene expression was maximum in the earlier and later stages of osteogenesis and adipogenesis, respectively. While PTHrP (107-139) did not modify the PTH1R overexpression during adipogenesis, PTHrP (1-36) did inhibit it; an effect which was partially affected by PTHrP (7-34), a PTH1R antagonist, at 1 microM. These findings demonstrate that both PTHrP domains can exert varying effects on human MSCs differentiation. PTHrP (107-139) showed a tendency to favor adipogenesis, while PTHrP (1-36) induced a mild osteogenic effect in these cells, and inhibited their adipocytic commitment. This further supports the potential anabolic action of the latter peptide in humans.

Evolution of PTH assays / Evolução dos ensaios para paratormônio

PTH metabolism is complex and the circulating forms include the intact 1-84 molecule as well as several carboxyl-terminal fragments. The first generation of PTH assays included several types of competitive assays, with specificities that spanned carboxyl, mid-region and amino-terminal portions of the molecule. The limitations of these assays and the methodological evolution led to the description of 2nd generation non-competitive immunometric assays for PTH in the late 80's, based on the recognition of the PTH molecule by two different antibodies, one directed against de amino-terminal and other against the carboxyl-terminal segments. The observation that in some circumstances "long" carboxyl-terminal segments were also measured by 2nd generation assays led to the development of 3rd generation assays based on amino-terminal specific antibodies that are specific for the first amino acids, measuring only the molecular forms that activate PTH1R. The practical and cost-benefit advantages of these assays are still debatable. The recent observation that carboxyl-terminal fragments of PTH have biological activity via a distinct receptor than PTH1R, points to the future need of more than one assay in order to evaluate parathyroid hormone function.

O metabolismo do PTH e complexo e as formas circulantes incluem o PTH 1-84, assim como fragmentos C-terminal. A primeira geração de ensaios para o PTH incluía vários ensaios competitivos com especificidades para as regiões carboxi, meio da molécula e amino-terminal. A limitação destes ensaios e a evolução metodológica, levaram ao desenvolvimento dos ensaios não competitivos de 2ª. geração no final dos anos 80, baseados no reconhecimento por dois anticorpos diferentes, contra a porção amino e carboxi-terminal respectivamente. A observação que em algumas circunstâncias segmentos carboxiterminais longos também eram detectados, levou ao desenvolvimento dos ensaios de 3ª. geração, baseados em anticorpos específicos para a porção aminoterminal com maior especificidade para os primeiros aminoácidos, e assim mensurando apenas a forma molecular que ativa o PTH1R. As vantagens práticas e o custo-benefício deste ensaio ainda e motivo de debate. A observação recente de que fragmentos carboxiterminais têm atividade biológica via receptor distinto, aponta para a necessidade futura de mais de um ensaio para avaliar a função do paratormônio.

Structure and Function of a Minimal Receptor Activation Domain of Parathyroid Hormone

The structure and function of short-length amino terminal PTH analogues were studied. The substitution of Leu7 with Phe in [Ala3, 10Leu7Arg11]rPTH (1-11) NH2 analogue peptides did not show any reduction in cAMP formation. Replacement of the 1st, 7th and 8th residues revealed different activities, depending upon the residue type. The substitution of Ala1 by Ser in [Ala3, 10Leu7Arg11]rPTH (1-11) NH2 caused nearly a complete loss of cAMP formation. Meanwhile, NMR analysis of [ (Ala1/ Ser1) Ala3, 10 (Leu7/Phe7) Arg11]rPTH (1-11) NH2 revealed an alpha- helical backbone structure with a flexible conformation at the carboxyl-terminus. The overall results suggest that 11-residue short oligopeptide analogues of PTH tend to form an alpha-helical structure and the different activities of those analogues could be associated with residue specificity rather than the secondary conformational structure.

Potential regulation of PTH/PTHrP receptor expression in choriocarcinoma cells

Parathyroid hormone-related peptide [PTHrP] have been found to be expressed in a variety of human tumors. Parathyroid hormone-related peptide is known as the major mediator of humoral hypercalcemia of malignancy, may also regulate placental calcium flux, uterine contraction and fetal tissue development. The purpose of this study is to evaluate the expression of PTH/PTHrP receptor in choriocarcinoma JAR cell line. This study was carried out at the Department of Biochemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia, between November 2002 and August 2003. Choriocarcinoma JAR cell line treated for 12 and 72 hours with epidermal growth factor, [EGF] [20ng/ml], estradiol, E2 [10-8 M], dexamethasone, [DEX] [10-8 M] or 1,25 dihydroxycholecalciferol, 1,25 [DHCC] [10-8 M]. We investigated the expression of parathyroid hormone [PTH]/PTHrP receptor in JAR cell line with these treatments compared with untreated JAR cells. The PTH/PTHrP receptor expression were detected with 3.3nM 125I-PTHrP-34Tyrosine. The expression of the receptors at 12 hours were increased following exposure to EGF, E2 or DEX, whereas 1,25 DHCC inhibited the receptor expression. In further experiments at 72 hours with the same treatments, the receptors expression were remarkably increased with EGF, E2 or DEX, whereas, 1,25 DHCC inhibited the receptor expression in these cells. These data suggested that in JAR cells, The EGF, E2 and DEX upregulated the PTH/PTHrP receptor expression, whereas the 1,25 DHCC down-regulated the PTH/PTHrP receptor, and the 1,25 DHCC may play an important role as antiproliferative drug for choriocarcinoma